Abstract

Despite the use of radiation and chemotherapy, the prognosis for children with diffuse brainstem gliomas is extremely poor. There is a need for relevant brainstem tumor models that can be used to test new therapeutic agents and delivery systems in pre-clinical studies. We report the development of a brainstem-tumor model in rats and the application of bioluminescence imaging (BLI) for monitoring tumor growth and response to therapy as part of this model. Luciferase-modified human glioblastoma cells from five different tumor cell sources (either cell lines or serially-passaged xenografts) were implanted into the pontine tegmentum of athymic rats using an implantable guide-screw system. Tumor growth was monitored by BLI and tumor volume was calculated by three-dimensional measurements from serial histopathologic sections. To evaluate if this model would allow detection of therapeutic response, rats bearing brainstem U-87 MG or GS2 glioblastoma xenografts were treated with the DNA methylating agent temozolomide (TMZ). For each of the tumor cell sources tested, BLI monitoring revealed progressive tumor growth in all animals, and symptoms caused by tumor burden were evident 26-29 days after implantation of U-87 MG, U-251 MG, GBM6, and GBM14 cells, and 37-47 days after implantation of GS2 cells. Histopathologic analysis revealed tumor growth within the pons in all rats and BLI correlated quantitatively with tumor volume. Variable infiltration was evident among the different tumors, with GS2 tumor cells exhibiting the greatest degree of infiltration. TMZ treatment groups were included for experiments involving U-87 MG and GS2 cells, and in each case TMZ delayed tumor growth, as indicated by BLI monitoring, and significantly extended survival of animal subjects. Our results demonstrate the development of a brainstem tumor model in athymic rats, in which tumor growth and response to therapy can be accurately monitored by BLI. This model is well suited for pre-clinical testing of therapeutics that are being considered for treatment of patients with brainstem tumors.

Comparison of tumor morphology associated with different GBM cell lines following implantation into the rodent pons. a 1 × 105 luciferase-modified U-87 MG, U-251 MG, GBM6, GBM 14, and GS2 glioblastoma cells were injected into brainstem in athymic rats using an implantable guide-screw system. b GS2 cells showed the greatest degree of infiltration within the pons, but show a lesser extent of infiltration than is evident following supratentorial injection in athymic mice

Bioluminescence monitoring of U-87 MG brainstem tumor xenografts treated with TMZ. a U-87 MG tumor bearing rats were treated with a daily dose of 50 mg/kg of TMZ or control vehicle delivered by oral gavage for 5 consecutive days beginning on day 15. Bioluminescence measurements for each rat were normalized against corresponding readings obtained at the beginning of therapy. TMZ treatment group (n = 4, white circle) shows a decreased normalized luminescence value through day 42, in contrast to the increasing luminescence value of control group, that was first evident at day 19 imaging (n = 5, black circle). b TMZ treatment significantly prolonged median survival (P = 0.0033) from 27 days in the control group (n = 5, black circle) to 53.5 days in the TMZ treatment group (n = 4, white circle). c TUNEL staining reveals an increase in the number of TUNEL-positive cells in tumor from the rat treated with TMZ, as compared with the tumor from the control animal (100× magnification; mean value = 26 vs. 0.75)

Bioluminescence monitoring of GS2 brainstem tumor xenografts treated with TMZ. a GS2 tumor bearing rats were treated with a single dose of 100 mg/kg of TMZ or control vehicle delivered by oral gavage on day 28. Bioluminescence measurements for each rat were normalized against corresponding readings obtained at the beginning of therapy. TMZ treatment group (n = 4, white circle) shows decreasing normalized luminescence until no signal is evident in treated mice, whereas control group mice show steadily increased luminescence from day 26 onward (n = 5, black circle). b TMZ treatment significantly prolonged median survival (P < 0.0001) from 44 days in the control group (n = 4, black circle) to >72 days in the TMZ treatment group (n = 4, white circle), at which time all TMZ treatment group rats were sacrificed and determined as being devoid of viable tumor